Intracellular adenosine regulates epigenetic programming in endothelial cells to promote angiogenesis

Abstract The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine‐metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro‐angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation‐specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK‐deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation‐induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.

Results are from three independent experiments.
For A-E, cells were treated with vehicle or adenosine at 10 µM for 24 hours. Adenosine treatment was repeated every 6 hours. Data information: For all bar graphs, data are the Mean ± SD, * P < 0.05 and ** P < 0.01 (Unpaired, two-tailed Student's t-test). The exact P-values are specified in Appendix Table S1.
Source data are available online for this figure.
Appendix Table S1. Statistical analysis information. Appendix Table S2. Pro-angiogenic genes with decreased methylation upon ADK knockdown.
Following the Infinium methylation assay, DNA methylation analysis was carried out on a group of 109 genes related to pro-angiogenic function as defined in the program of Gene Set Enrichment Analysis (GSEA). Column 1: gene symbols and names according to the HUGO Gene Nomenclature Committee; column 2: gene accession number according to the National Center for Biotechnology Information; column 3: number of different probes for each gene represented on the array; column 4: CH 3 ↑, number of probes with increased methylation in the shADK group compared with the shctl group; column 5: CH 3 ↓, number of probes with reduced methylation in the shADK group compared with the shctl group; column 6: CH 3 ↑, percentage of probes with increased methylation in the shADK group compared with the shctl group; column 7: CH3↓, percentage of probes with reduced methylation in the shADK group compared with the shctl group. The level of DNA methylation in the 14 green highlighted pro-angiogenic genes are considered to be significantly decreased in the shADK group compared with the shctl group.
Appendix Table S3: Anti-angiogenic genes with increased and decreased methylation upon ADK knockdown. Appendix Table S3. Anti-angiogenic genes with decreased methylation upon ADK knockdown.
Following the Infinium methylation assay, DNA methylation analysis was carried out on a group

Mouse generation and breeding
The

Isolation and culture of primary mouse aortic endothelial cells (MAECs)
MAECs were isolated using a previously described method except that Matrigel was replaced with collagen gel (Wang et al, 2016). Collagen gel was prepared as follows.
Commercial type I collagen (BD Bioscience, San Jose, CA, USA) was diluted with endothelial growth medium 2 (EGM-2; Lonza, Basel, Switzerland) to a final concentration of 1.75 mg/mL. 0.5 ml collagen gel was added to each well of a 24-well plate, and the collagen gel was then solidified at 37°C for at least 30 min.
Aortic ring preparation: Four-week-old male mice were used. Briefly, mice were sacrificed by CO 2 asphyxiation and cleaned with 70% ethanol. The abdominal and thoracic cavities were opened, and each mouse was perfused with 3 mL PBS via the left ventricle. After removal of perivascular fat and adventitia from the ventral side of the aortas, the aortas were dissected out, rinsed 5 times with fresh PBS, and placed in a sterile dish of cold PBS. The aortas were then cut into ~1 mm length rings, and each aortic ring was opened and laid on the surface of collagen gel with the endothelium directly facing the gel.
Following tissue placement for 36 hours, the gel and aortic piece were kept hydrated with EGM-2. During the above procedures, care was taken to avoid submerging and dislodging the aorta piece from the gel. Explants were then cultured in an incubator at 37°C and 5% CO 2 and monitored daily. For the experiments on endothelial migration, the gels and aortic pieces were processed at different time points based on the protocols. After visible cellular outgrowth from the aortic segments, the medium was aspirated, and the aortic segments were removed in a sterile fashion. The collagen gel was digested by 0.3% collagenase H solution in PBS, and the MAECs were collected.

Isolation and culture of murine lung and heart endothelial cells
Lungs and hearts were minced and placed in DMEM containing 2 mg/ml collagenase (Roche, Basel, Switzerland) for 1 h at 37 o C. The digested tissues were separated into single cell suspension using 18-gauge and 20-gauge needles and then passed through a 70 µm cell strainer.
The cell suspension was centrifuged and the cell pellets were washed once with washing buffer

Adenoviral transduction of HUVECs
The GFP-labeled-ADK shRNA adenovirus targeting the 3' UTR sequence of human ADK and the control adenovirus were constructed by Vector Biolabs (Malvern, PA, USA). Ad-mutHIF-1α encoding the mutant human HIF-1α construct containing mutations at P564A and N803A and Ad-mutHIF-2α containing mutations at P531A and N847A were generated as previously described (Ahmad et al, 2009). These adenoviruses were expanded inside HEK293 cells, and the virus concentration was determined using an Adeno-XTM rapid titer kit (Clontech, Mountain View, USA). HUVECs at 80% confluence were transduced with the adenovirus (10 pfu/cell) and were used for experiments 36 h after the transduction.

RNA interference
HUVECs were transfected at 60-70 % confluence with 30 nM siRNAs targeting human

Cell number analysis
For cell counting, HUVECs or MAECs were seeded in 6-well plates in triplicate at an equal density, and cell numbers were manually counted at the indicated days with a hemocytometer.

Quantitative real time RT-PCR (qRT-PCR) analysis
The total RNA from HUVECs was extracted with an RNeasy Mini Kit (Qiagen), and qRT-PCR was done as described previously. Briefly, a 0.5-1 μg sample of RNA was utilized as a template for reverse transcription using the iScriptTM cDNA synthesis kit (Bio-Rad). qRT-PCR was performed on an ABI 7500 Real Time PCR System (Applied Biosystems) with the respective gene-specific primers listed in Appendix Table S4. All samples were amplified in duplicate, and every experiment was repeated independently at least three times. For experiments under normoxia, relative gene expression was calculated using the 2 - ct method against the internal control β-actin. For experiments subjected to hypoxia, relative gene expression was calculated using the 2 - ct method against the internal control RPLP0.

Protein extraction and Western blotting
HUVECs or MAECs were lysed with RIPA buffer (

In vitro tube formation analysis
HUVECs were transfected with an ADK shRNA-containing adenovirus or control virus 36 h before the assays were performed, or cells were treated with 10 μM adenosine 48 h before assays were performed. To examine tube formation, growth factor-reduced Matrigel (BD Bioscience, San Jose, CA, USA) was placed in 96-well tissue culture plates (60μl/well) and allowed to form a gel at 37°C for at least 30 min. HUVECs infected with virus were resuspended in 0.5% FCS growth medium at a 1 × 10 5 concentration. The adenosine-treated HUVECs were resuspended in 0.5% FCS growth medium supplemented with 10μM adenosine.
Aliquots of 150 μl of the cell suspension were added to each well, and the plates were incubated Observer Z1 inverted microscope at 10 X magnification.

Fibrin gel bead assay
Fibrin gel bead assay was performed as described previously (Nakatsu et al, 2007). Briefly, HUVECs were incubated with Cytodex3 microbeads (Amersham Pharmacia Biotech) at a concentration of 400 cells per bead in 1.5 mL of EGM-2 medium for 4 hours at 37°C. Overnight, cell-coated beads were washed with EGM-2 and then resuspended in 2 mg/mL of fibrinogen solution (Sigma-Aldrich) plus 0.15 U/mL of aprotinin (Sigma-Aldrich) at a concentration of 500 cell-coated beads/mL. Next, 500 μL of fibrinogen/bead suspension was added to the wells of a 24-well plate containing 0.625 units of thrombin (Sigma-Aldrich). Once the gel clotted, 1 ml of EGM2 containing 20,000 fibroblasts was placed on the top of the fibrin gel, and the culture medium was changed every other day. Images were captured and data were quantified between days 5 and 6 by the live-culture imaging system of a Zeiss LSM 780 Inverted Confocal Microscope using a bright field. The number of sprouts and cumulative length of sprouts per bead were quantified from 30 beads per condition.

Spheroid capillary sprouting assay
HUVECs (750 cells) were incubated for 24 hours in 25% EBM-2 (25% EBM-2 complete medium plus 75% EGM-2 medium) supplemented with 0.25% (w/v) methylcellulose (Sigma-Aldrich) to form spheroids as described previously (Heiss et al, 2015). The spheroids were harvested and embedded in 0.9 ml collagen solution in a pre-warmed 24-well plate, with a final concentration of rat type I collagen (BD Biosciences) at 1.5 mg/ml. The spheroid-containing gels were rapidly transferred into a humidified incubator (37 °C, 5 % CO2) and allowed to polymerize for 20 min, after which 0.1 ml EBM-2 basal medium supplemented with 20ng/ml hVEGF (R & D Systems, Minneapolis, MN, USA) and the corresponding compounds were pipetted on top of the gel. After 24 hours, gels were fixed with pre-warmed 4 % paraformaldehyde (PFA), and images were captured with a Zeiss LSM 780 Inverted Confocal Microscope. The number of sprouts per spheroid was quantified from 10 spheroids for each condition using Image J software.

Aortic ring assay
Aortas were harvested from 4-week-old male ADK VEC-KO and control ADK WT mice.
Plates (24-well) were coated with collagen gel, and after it had gelled, the rings were placed into the wells. Thirty-six hours later, 1 mL EGM-2 was added to each well. On day 3 or 5, the tissue and the sprouting endothelial cells were fixed and stained with isolectin B4. Endothelial sprouts were time-lapse imaged on an Olympus IX81-ZDC inverted fluorescence microscope (Olympus, Center Valley, PA, USA). Vessel sprouting was quantified by counting the number of vascular sprouts that directly originated from a mouse aorta. The photographed image was divided into 5 regions and sprouts in each region were scored from 0 (least positive) to 1 (most positive) in a double-blind manner. The scores from 5 regions were combined, and the total was scored from 0 (least positive) to 5 (most positive). Each data point was assayed in quintuple.

Analysis of mouse retinal vasculature
For retinal vessel analysis, P4 or P12 pups were given lethal doses of ketamine Jena, Germany). Vessel sprouts were evaluated using a described method (Lobov et al, 2007).

Analysis of embryonic hindbrain angiogenesis
Mouse embryos were harvested at E10.5-11.5 and hindbrain tissue was isolated, stained and analyzed as previously described (Fantin et al, 2013).

Wound healing assay and analysis
A previously described method was used (Lanahan et al, 2014). Briefly, 8-week-old male ADK VEC-KO and control ADK WT mice were anesthetized by intraperitoneal injection of a ketamine (100 mg/kg)/xylazine (10 mg/kg) solution. Wounds were generated with a sterile 6-mm biopsy punch (Miltex Inc, PA) on the mouse back skin without injuring the underlying muscle.
Wound regions were photographed using a Leica M125 microscope with an HC80 HD camera (Leica, Wetzlar, Germany) on days 0, 1, 3, 5 and 7. The wound area was calculated using NIH Image J software in a blinded fashion with regard to the animal groups, and the sizes at different time points were expressed as a percentage of the wound area on day 0. Blood perfusion images of the wounds on day 2 of wound generation were obtained by laser speckle contrast imaging.
Briefly, the mice were anesthetized using isoflurane, and body temperature was maintained at 37 ±0.2 °C using a thermo-regulated heating pad (Harvard Apparatus, Holliston, MA, United States).
Blood perfusion images were acquired using PeriCam PSI HD system (Perimed Inc., Sweden) with a 70 mW built-in laser diode for illumination and a 1388 x 61038 pixel CCD camera for image acquisition. Once the perfusion was consistent, images (2 per sec) were acquired for 2 min at a speed of 2 Hz. Acquired images were analyzed for blood flow using PIMSoft, a PeriCam dedicated computer program (Perimed Inc., Sweden). The mean wound blood flow was calculated as the ratio of the blood flow in the wound area to that of the surrounding normal tissue area and was presented as a percent. Six mice were analyzed in each group.

Hind limb ischemia model
As described previously (Limbourg et al, 2009), the femoral arteries of male ADK VEC-KO and control ADK WT mice at 8 weeks of age were ligated at 2 positions spaced 5 mm apart, and the arterial segment between the ligatures was excised. In a blinded fashion with regard to the animal groups, tissue perfusion was assessed preoperatively, immediately, and 3, 7, and 14 days after surgery. Blood flow images of the foot were acquired by laser speckle contrast imaging (Perimed Inc., Sweden) at 37 ± 0.2 °C under isoflurane anesthesia. Data were analyzed with PIMSoft, a PeriCam-dedicated computer program (Perimed Inc., Sweden) and reported as the ratio of blood flow in the left/right (L/R) hind limb after background subtraction.

Histology and immunohistochemistry
On the day indicated after femoral artery ligation, gastrocnemius muscles were dissected, fixed in 4% PFA for 24 h, dehydrated, embedded in paraffin, sectioned at 7 μm thicknesses, and stained with haematoxylin and eosin (H&E). Areas of necrotic muscle fibers were identified with morphology, differential eosin staining, and infiltrating leukocytes near the degenerating fibers on the whole section at × 200. Inflammation status was scored based on the number of infiltrated leukocytes.
For immunofluorescent staining of CD31 and VEGFR2 in gastrocnemius muscles, following deparaffinization, rehydration and antigen retrieval, sections were blocked and incubated overnight at 4°C with primary antibodies followed by fluorescent secondary antibody

Global DNA methylation assay
Total genomic DNA was isolated from HUVECs using a DNeasy Blood and Tissue Kit (QIAGEN, Venlo, Netherlands). Global DNA methylation status was assessed using the MethylFlash Methylated DNA quantification kit (Epigentek, Farmingdale, NY, USA) per the manufacturer's instructions.

DNMT activity assay
Nuclear proteins were isolated from adenosine-treated HUVECs using an EpiQuik Nuclear Extraction Kit I per the manufacturer's instruction (Epigentek, Farmingdale, NY, USA).
DNMT activity of freshly isolated nuclear proteins was quantified using a fluorimetric EpiQuick

Infinium methylation assay
The bisulfite conversion of genomic DNA was conducted using an EZ DNA Methylation

Measurement of intracellular cAMP
The cAMP concentrations were determined using a Cyclic AMP XP® assay kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's protocol. Briefly, HUVECs were seeded at a density of 1 × 10 4 cells/well in a 96-well plate and incubated overnight. Following exposure to the indicated chemicals, the cells were washed with ice-cold PBS and lysed with ice-cold 1× lysis buffer. The HRP-linked cAMP solution was added to the assay plate, incubated at room temperature for 3 h, followed by 4 washes with the 1× wash buffer. The TMB substrate was then added and the absorbance was measured at 450 nm. The cAMP concentration of the sample was calculated using a cAMP standard curve.

Measurement of intracellular adenosine, SAM and SAH levels
Adenosine was measured with reversed-phase HPLC. Briefly, supernatants of HUVEC lysates with protein concentration at 1 mg/mL were separated with a C 18 reversed-phase analytical column (250 mm x 4.6 mm I.D., 5 μm particle) (Aglient, Santa Clara, CA, USA). The mobile phase consisted of two solvents. Solvent A is a solution of 0.031M Na 2 HPO 4 ·2H 2 O and 0.068M NaH 2 PO4·2H 2 O adjusted to pH 6.3. Solvent B is a solution of 100% methanol. Solvent A was filtered through a 0.2-μm membrane filter prior to use in the assay. To detect adenosine, the HPLC column was first equilibrated with 80% Solvent A and 20% Solvent B and was held constant at the equilibration conditions for 10min. The flow-rate was 1 mL/min, and detection was monitored at 254 nm. A 20-μl aliquot of the acid extract was applied directly onto the HPLC column. The identity of adenosine was determined by comparing retention times to an adenosine standard and was further confirmed by enzymatic peak shift analysis. The results (µm/mg) indicate the adenosine concentrations (µmol/L) in the cell lysates with protein concentration at 1 mg/mL. SAH and SAM were measured with reversed-phase HPLC. Briefly, supernatants of HUVEC lysates were separated with a C 18 reversed-phase analytical column (250 mm x 4.6 mm I.D., 5 μm particle) (Aglient, Santa Clara, CA, USA). The mobile phase consisted of two solvents. Solvent A is a solution of 8 mM octane sulfonic acid sodium salt and 50 mM NaH 2 PO 4 adjusted to pH 3.0 with H 3 PO 4 . Solvent B is a solution of 100% methanol. Solvent A was filtered through a 0.2-mm membrane filter before use in the assay. The HPLC column was first equilibrated with 80% Solvent A and 20% Solvent B and then injected with sample. Separation was obtained using a step gradient with 8 min at the equilibration conditions, 30 s to increase Solvent B to 40%, 12.5 min at the new condition, and 30 s to return to the equilibration conditions. There was a minimum of 10 min before a subsequent injection. The flow-rate was 1 ml/min, and detection was monitored at 254 nm. A 25-μl aliquot of the acid extract was applied directly onto the HPLC column. SAM and SAH were identified based on their retention times and co-chromatography of SAM and SAH standards. Quantification was based on integration of peak areas and compared to the standard calibration curves of SAM and SAH.

Statistical analysis
The data are presented as the mean ± SD and were analyzed by one-way ANOVA followed by Tukey's post-hoc test or Student's t-test to evaluate two-tailed levels of significance.
Two-way repeated-measures ANOVA with Bonferroni's post-hoc test was done to assess wound size and perfusion improvement over time within groups. The null hypothesis was rejected at P ≤ 0.05.